Sealed disks isolated from bovine retinal rod outer segments by hypotonic lysis and centrifugation on a Ficoll gradient were characterized in terms of their membrane proteins and sideness. High resolution SDS-polyacrylamide slab gel electrophoresis indicated that, in addition to the major protein rhodopsin, nine minor proteins ranging in molecular weights from 39,000 to 250,000 were present in these preparations. Fluorescent lectin labeling studies of proteins fractionated on SDS-gels indicated that both rhodopsin and a 250,000 molecular weight component designated as ROS 1.2 were glycoproteins having concanavalin A (ConA) and wheat germ agglutinin (WGA) binding sites. The sideness of the sealed disks was studied using 125I-ConA. Results indicated that no significant binding of 125I-ConA was observed unless the disks were subjected to 0.03% Triton X-100 or several freeze-thaw cycles. This suggests that the carbohydrate residues of rhodopsin and ROS 1.2 are oriented toward the intradisk space in sealed disks where they are inaccessible to ConA binding. Proteolysis of sealed disks, however, resulted in the degradation of rhodopsin and ROS 1.2 into smaller membrane bound fragments. These studies support the view that both rhodopsin and ROS 1.2 span the disk membrane.